This application is in response to RFA (MH-97-001) "Innovative Approaches for Microscopic Tract-Tracing." The proposed studies involve the collaboration of researchers within the Neurosciences Research Center, Allegheny University of the Health Sciences and the Center for Light Microscopic Imaging and Biotechnology, Carnegie Mellon University. Collectively, the key personnel bring to these studies many years of experience with neuroanatomy, synthetic chemistry, membrane biology, and microscopic imaging. Moreover, the work of the Principal Investigator on Alzheimer's disease (AD) provides an experimental framework with which to develop novel tracing compounds and to investigate disease-related alterations in brain circuitry. In this application we propose a series of independent yet highly correlated studies. These include: Aim 1: the identification and evaluation of fluorescent molecules which rapidly diffuse through the lipid membranes of aldehyde-fixed human post-mortem material. It is our hypothesis that the molecular weight and chemical structure of a compound will determine its interaction with the membrane and hence the mobility of the compound. Aim 2: characterization of the molecular interactions of tracer dyes with aldehyde-fixed membranes using small-angle x-ray scattering approaches. Our hypothesis states that dyes will partition to discrete sites in the lipid bilayer as a function of their amphophilic properties and membrane composition. Aim 3: development and evaluation of active novel transport techniques for enhancing the retro- and anterograde movement of tracing compounds. Preliminary studies support the use of electrical currents to enhance the migration of dyes. Aim 4: development of lipid tracer dyes that are compatible with the processing of tissue sections for conventional histological and immunohistochemical techniques. Aim 5: development of procedures to simultaneously visualize multiple novel tracing dyes in fixed tissue. The application of the novel tracer dyes and methods developed in Aims 1-5 will be applied to the study of the human hippocampus of aged matched controls and subjects with AD (Aim 6). It is our hypothesis that subjects with AD will display marked alterations in the microcircuitry of anatomical structures known to subserve memory and cognition (e.g., hippocampus).